Evaluation of morphological traits, biochemical parameters and seeding availability pattern among Citrus limon ‘Assam lemon’ accessions across Assam

The Assam lemon is a highly valued Citrus cultivar known for its unique aroma, flavor, and appearance. This study aimed to investigate the morphological, seeding pattern and biochemical variations within 132 populations of Assam lemon from across 22 districts of Assam along with the control samples, with the objective to offer comprehensive understanding that could facilitate the improvement of breeding programs and further improvement of this important cultivar. Clustering based on UPGMA algorithm for morphological and seeding pattern data were analysed at population level, revealed two major clusters, where all the populations of Upper Assam districts were in the same cluster with the original stock (control population). The populations from Tinsukia and Dhemaji districts displayed more close similarities with the control population in comparison to populations of Upper Assam districts. Another interesting observation was regarding flowering patterns, while populations from Upper Assam districts excluding Golaghat district displayed both bisexual and unisexual flowers with less concentration of unisexual flowers, other remaining districts had bisexual and unisexual flowers of almost equal concentration. Unisexual flowers contained only the male reproductive organs with 40 anthers, while bisexual flowers had 36 anthers. Seeding patterns were examined across the districts, and it was found that populations from Tinsukia, Dhemaji, Lakhimpur, Dibrugarh, Jorhat, and the control population exhibited seedless characteristic while populations from other selected districts displayed a combination of seedless and seeded traits. Interestingly, Golaghat district appears as the linking district and showed availability of both seeded and seedless Assam lemon fruit, connecting the regions of Barak valley, Central, Lower, North and Upper Assam. Biochemical analysis showed significant variations across districts, however, the populations from Dhemaji, Tinsukia, Lakhimpur, Dibrugarh, and Jorhat districts displayed similarity with the control population. The study also investigated variability in soil nutrient content revealing substantial variation among the populations studied. This comprehensive investigation provides valuable insights into the morphological, seeding pattern, and biochemical diversity within the Assam lemon cultivar. These findings can be instrumental in breeding programs to enhance the cultivar, particularly in producing high-quality seedless fruits to meet consumer demands.


Fruit materials
Fresh fruit samples of Assam lemon were systematically collected from a total of 132 populations across 22 selected districts in Assam (Supplementary file 1).Additionally, samples were also obtained from the Horticulture Research Station, Kahikuchi, which served as the control population for comparison (Supplementary file 1).These collected fruit samples were carefully handled and stored in a refrigerated environment at − 80 °C until the completion of the present investigation.

Morphological analysis
For our research on morphological diversity across the various districts of Assam, we carefully considered a comprehensive set of 70 variables.These variables encompass a wide range of characteristics related to trees, including their physical traits, leaves, flowers, fruits, seeds etc. and the observations were recorded during the time of the investigation.To ensure a thorough analysis, we examined the following aspects which includes, tree character, tree height, branches density, branches angle, thorn density, thorn size, thorn shape, leaf density, leaf division, leaf venation, leaf shape, leaf apex, leaf margin, leaf intensity of green, leaf width, leaf length, flower nature, flower sexuality, flower color, flower length (cm), sepal arrangement, sepal color, sepal length (cm), petal arrangement, petal fusion, petal adaxial side color, petal abaxial side color, petal length (cm), androecium arrangement, androecium length (cm), number of anthers, anther arrangement, anther color, anther length (cm), filament color, filament length (cm), gynoecium arrangement, gynoecium length (cm), sigma color, stigma length (cm), style color, style length (cm), ovary color, ovary length (cm), % flowering shoot, flower density, % full bloom flower, fruit density, fruit color, fruit shape, fruit skin texture, fruit base, fruit apex, fruit axis, fruit weight, fruit length, fruit diameter, specific gravity, number of segments, fruit peel weight, fruit pulp weight, fruit pulp : peel ratio, fruit rind thickness (mm), albedo thickness (mm), flavedo thickness (mm), the length from pith to albedo (mm), pith diameter (mm), seed availability, seed number, seed color.The data collected was further analyzed using statistical techniques and clustering methods, to identify patterns, relationships, and potentially distinct groups of Assam lemon within the studied regions.

Seeding pattern
For the investigation of seeding pattern of Assam lemon, mature fruits were sampled from different populations of 22 districts throughout the Assam.In addition, fruits were collected from the Horticulture Research Station, Kahikuchi to serve as a control population for comparison.The mature fruits were carefully collected at their maturity stage (60 DAF) to ensure accurate seed analysis.In the laboratory, the collected fruits were gently washed and dried to remove any external contaminants.Subsequently, each fruit was cut open, and the seeds were carefully extracted using a sterile knife, and forceps.The total number of seeds per fruit was counted, and their color was recorded.The data obtained from the seed analysis were subjected to statistical analysis using methods described below to determine the average number of seeds per fruit and assess any significant variations among different samples (Supplementary file 1).

Biochemical analysis
The fruit quality characteristics of selected districts of Assam along with control population were assessed, focusing on several parameters (Supplementary file 1).These parameters included pH level, % of juice content, total soluble solids (measured in °Brix), citric acid concentration (measured in g/ml), ascorbic acid concentration (measured in mg/ml), total soluble solids to titratable acidity ratio, total sugar content (measured in µg/ ml), reducing sugar content (measured in µg/ml), carotenoid concentration (measured in mg/g), chlorophyll content (measured in µg/g), pectin content (both in the peel and pulp), equivalent weight (for both peel and pulp), methoxy content (for both peel and pulp), anhydrounic acid content (for both peel and pulp), and degree of esterification (for both peel and pulp).
pH Total pH of the Assam lemon juice were measured using pH 700 m (Eutech Instruments, United Kingdom) 26 .

Total soluble solids
Total soluble solids of the lemon juice were determined as °Brix using OPTi digital refractometer (Bellingham + Stanley, United Kingdom) 27 .
% juice content % juice content determination was performed following the methodology described by Kashyap et al. in 2020, with slight modifications 28 .The weight of both the fruit and its corresponding juice content were measured in grams and documented.The % of juice content was then calculated using the formula given below,

Citric acid
The determination of citric acid content in the juice of Assam lemon fruits was done using a modified protocol described by Brima et al. in 2014 29 .To measure the concentration of citric acid, the lemon juice was extracted by squeezing the fruits and then diluted with distilled water at a ratio of 1:9 (Juice:Water).Phenolphthalein was added as an indicator, and the titration was conducted using NaOH as the base.The titration process continued until the color of the juice turned red/pink and remained consistent for at least 15 s.This titration was repeated with additional aliquots of the sample solution until concordant results were obtained.The concentration of citric acid was then calculated using the formula given below, where A = Mean value of coordinate readings.Acid factor = 0.0064.

Ascorbic acid
The determination of ascorbic acid content in the juice of Assam lemon fruits, a modified protocol described by Satpathy et al. in 2021 was followed 30 .To measure the concentration of ascorbic acid, 20 ml of fresh lemon juice filtrate was transferred into a 250 ml conical flask, and 1 ml of starch indicator was added.The sample solution was then titrated against a 0.01 mol L −1 iodine solution until the color changed to a dark black color.The titration process was repeated with additional aliquots of the sample solution until concordant results were obtained.The concentration of ascorbic acid was then calculated using the formula given below,

Total soluble solids/titratable acidity (TSS/TA)
The TSS/TA ratio of Assam lemon fruit was assessed using the methodology outlined in the study conducted by Kashyap et al. in 2020 28 .This ratio was obtained by dividing the °Brix value (representing the Total Soluble Solids) by the percentage of acid content.

Total sugar and reducing sugar
The quantification of reducing sugar was performed using 3,5-dinitro salicylic acid (DNS), following the modified protocol described by Gusakov et al. in 2011 31 .Additionally, the determination of total sugar was conducted using Anthrone reagent, as outlined by Buckan in 2015 with slight modifications 32 .

Carotenoid content
The carotenoid content of the peel samples from Assam lemon was determined using the methodology described by Kashyap et al. in 2020, with slight modifications 28 . 1 g sample of fruit was ground with liquid nitrogen, and the resulting powder was mixed with 10 ml of a hexane:acetone:ethanol solution (v/v; 50:25:25).Subsequently, the mixture was centrifuged at 4000 g for 5 min.The supernatant, containing the color compounds, was collected, and the volume was adjusted to 10 ml with the extraction solvent.The total carotenoid content was then measured by assessing the absorbance at 450 nm using a spectrophotometer.The concentration of carotenoids was calculated using the following equation, where A = Absorbance at 450 nm wavelength.V = Total volume of sample.W = Weight of fresh plant tissue.

Chlorophyll content
The chlorophyll content of the juice of Assam lemon at various fruit developmental stages were determined using the protocol described by Kashyap et al. 2020 with minor modification 28 .For the determination of chlorophyll concentration in the fruit peel, 1 g of peel sample was ground with liquid nitrogen.The resulting powder was then mixed with 20 ml of 80% acetone solution containing 0.5 g of MgCO 3 .After incubating the mixture at 4 °C for 3 h, it was centrifuged at 2500 rpm for 5 min.The supernatant was carefully collected, and its absorbance was measured at 654 nm and 663 nm using a spectrophotometer.Using the provided equation, the concentration of chlorophyll was calculated.
where A = Absorbance at specific wavelength.V = Final volume of chlorophyll extract in 80% acetone.W = Fresh weight of tissue extracted.

Pectin content
The determination of pectin content in both the peel and pulp samples of Assam lemon was done following the protocol outlined by Khamsucharit et al. in 2017, with minor modifications 33 .A total of 5 g of dried fruit sample was mixed with 100 ml of Citric acid solution (prepared by mixing 9 parts distilled water with 1 part Citric acid) and incubated at 65 °C for 1 h.After the completion of incubation, the extract was filtered, and 95% ethanol was added to the filtrate in a 1:1 proportion.The mixture was then incubated at room temperature for 2 h.Following incubation, the flocculants were skimmed off and washed 2-3 times using ethyl alcohol.The resulting precipitate was thoroughly dried at 35-40 °C and weighed to determine the pectin concentration.The concentration of pectin was calculated using the provided equation.
where Ypec = Yield of pectin.P = Amount of extracted pectin.Bi = Initial amount of fruit powder.

Equivalent weight
The determination of equivalent weight content in both the peel and pulp samples of Assam lemon was done following the protocol outlined by Khamsucharit et al. in 2017, with minor modifications 33 .In this process, 0.5 g of dried pectin sample obtained from the previous pectin estimation experiment was dissolved in 5 ml of ethanol.
To this pectin solution, 1 g of NaCl and a few drops of phenol red indicator were added.The resulting mixture was titrated against 0.1 M NaOH until a pale pink color, indicating the end point, was achieved.The titration was www.nature.com/scientificreports/repeated with additional aliquots of the sample solution until consistent and concordant results were obtained.The equivalent weight was then calculated using the provided equation.

Methoxy content (MeO)
The determination of methoxy content in both the peel and pulp samples of Assam lemon was done following the protocol outlined by Khamsucharit et al. in 2017, with minor modifications 33 .The methoxy content was estimated using the neutralized titrated solution obtained from the equivalent weight estimation.To the solution, 0.25 M NaOH was added and stirred for 30 min, followed by the addition of 25 ml of 0.25 N HCl.The resulting mixture was titrated against 0.1 N NaOH until a pale pink color, indicating the end point, was achieved.The titration was repeated with additional aliquots of the sample solution until consistent and concordant results were obtained.The methoxy content was then calculated using the provided equation.

Anhydrounic acid (AUA)
The anhydrounic acid content in both the peel and pulp samples of Assam lemon was determined according to the method described by Khamsucharit et al. in 2017, with slight modifications 33 .The calculation of anhydrounic acid content was performed using the provided equation.
where Molecular weight = 176 g.Z = Equivalent weight titration result.Y = Methoxy content titration result.W = Weight of sample.

Degree of esterification (DE)
The degree of esterification in both the peel and pulp samples of Assam lemon was determined using the method described by Khamsucharit et al. in 2017, with slight modifications 33 .The calculation of the degree of esterification was carried out using the equation provided.

Statistical analysis
For statistical analysis, the numerical data was generated using morphological characters including seeding pattern and also biochemical attributes which was used for the statistical analysis during the current investigation 34,35 .Further, using the PAST 4.11 program, we performed, Principal Coordinate Analysis (PCoA), and constructed a dendrogram using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on these numerical data generated using morphological characters.Also, using the PAST 4.11 program, Analysis of Variance (ANOVA) test with post hoc Duncan Multiple Range Test (DMRT) was performed using SPSS 26 program to determine significant differences in morphological and biochemical attributes.

Soil nutrient estimation
Soil samples were carefully collected from various districts of Assam, focusing on locations with thriving Assam lemon trees during their active growth phase.Care was taken to avoid areas with apparent disturbances.Soil samples were collected to a depth of 15 cm, and several sub-samples were combined to create a composite sample at each site 36 .The collected soil samples were preserved and transported to the laboratory for analysis, where various physicochemical properties, including, pH, availability of macronutrients and micronutrients were measured.
To assess the soil conditions in the 22 districts of Assam along with the control population, we employed soil testing kits (K054-1KT and K095L-1KT, Himedia) (Supplementary file 1).These kits enabled us to estimate the concentrations of both micronutrients and macronutrients present in the soil samples collected for the current investigation.In terms of micronutrients, we focused on assessing the levels of Copper, Zinc, Boron, Manganese, Iron, and Molybdenum.These elements play crucial roles in plant growth and development, and their availability in the soil can significantly impact the health and productivity of vegetation.For macronutrient analysis, we evaluated several key parameters, including pH levels, organic Carbon content, Phosphate concentration, Potassium levels, Ammoniacal Nitrogen content, and Nitrate Nitrogen levels.These macronutrients are essential for plant nutrition, as they are involved in various physiological processes and influence overall soil fertility.This study complied with relevant institutional, national, and international guidelines and legislation of India, and no specific permits were required to collect the plant materials.The authentication of the collected samples was done by Department of Botany, Gauhati University under the guidance of curator Dr. Souravjyoti Borah.The voucher specimen of the collected plant samples has been deposited in the herbarium of Department of Botany at Gauhati University and the accession number GUBH19963 was issued for the same.

Equivalent Weight =
Weight of pectin samples × Molarity of alkali Volume of alkali × 100

Morphological analysis
The analysis of morphological characters in Assam lemon revealed significant variation across different regions of the state (Supplementary file 2, Supplementary file 3, Supplementary file 4).The study encompassed a total of 70 morphological characters, including tree characteristics, thorn characters, leaf traits, flower attributes, fruit properties, and seed features.Among all the studied characters, the flower traits exhibited the most pronounced variation.Notably, the investigation of Assam lemon flowers indicated that accessions from control, Dhemaji, Tinsukia, Jorhat, and Dibrugarh displayed both bisexual and unisexual flowers but, the concentration of unisexual flowers was comparatively quite less in these regions.In contrast, accessions from Golaghat, Central Assam (Karbi Anglong, Dima Hasao, Nagaon, and Morigaon), North Assam (Udalguri, and Sonitpur), Lower Assam (Kamrup Metropolitan, Kamrup Rural, Baksha, Nalbari, Barpeta, Bongaigaon, Kokrajhar, and Dhubri) to Barak valley (Dima Hasao, Cachar, and Karimganj) exhibited a combination of bisexual and unisexual flowers, having unisexual flowers of almost equal concentration to that of bisexual flowers.The unisexual flowers possessed only the androecium (Fig. 7).Furthermore, the study revealed a distinct difference in the number of anthers between unisexual and bisexual flowers.Unisexual flowers were found to have as much as 40 anthers, whereas bisexual flowers exhibited 36 anthers (Supplementary file 3).Additionally, significant variations in fruit morphological characters were also observed across 690 accessions of Assam (Fig. 8, Supplementary file 4).

Seeding pattern
During the investigation into the seeding pattern of Assam lemon across the 22 districts of Assam, an interesting finding emerged.It was observed that there is a combination of seeding patterns within the studied districts, with some districts exhibiting the true seedless trait characteristic of Assam lemon, while others exhibited a mixed character and had both seedless and seeded type (Fig. 9).Also, we have observed that in the study area of Upper Assam districts and control, the Assam lemon trees were cultivated as a standalone crop.However, in other selected districts, the Assam lemon trees were found to be cultivated as standalone or alongside other Citrus varieties.Throughout the current investigation, we observed that the fruits collected from Tinsukia, Dhemaji, Lakhimpur, Dibrugarh, and Jorhat districts including control population were seedless.However, during the current investigation, we found a mixture of seedless and seeded fruits in the populations of Golaghat district, districts of Central Assam, North Assam, Lower Assam and Barak Valley districts.Interestingly, it appears that Golaghat district serves as the linking district where both the seeded and seedless fruits are present connecting the regions of Central Assam, North Assam, Lower Assam and Barak valley (Fig. 10).Surprisingly, we discovered significant variation in the presence and quantity of seeds among the different districts of Central Assam, North Assam, Lower Assam and Barak Valley.Each district exhibited its own distinct seed count in the fruits where seeds were observed.While, studying the seed count of Assam lemon fruits, we made some interesting observations accessions from Golaghat, Sonitpur, Nagaon, Morigaon, and Nalbari districts consistently exhibited a similar pattern, with seed counts ranging from 0 to 5 seeds per district.On the other hand, when studying the accessions from Karbi Anglong, Udalguri, Baksha, Kamrup Metro, and Kamrup Rural districts, we found the seed counts ranged from 0 to 10 seeds per districts.Additionally, accessions from Barpeta, Bongaigaon, Kokrajhar, Dhubri, Dima Hasao, Cachar, and Karimganj districts also showed mixed seed counts, varying from 0 to > 10

PCoA analysis
A Principal Coordinates Analysis (PCoA) was conducted to visualize the spatial patterns of morphological variation among populations and districts of Assam lemon.The results of the analysis showed that the populations from districts of Dhemaji, Tinsukia, Dibrugarh, Lakhimpur, Jorhat, and Dima Hasao shared highest morphological similarities with the control population (Fig. 11).On the other hand, the populations from the districts of, Kamrup Metro, Kamrup Rural, Nalbari, Barpeta, Sonitpur, Nagaon, Morigaon, Baksha, Udalguri, Bongaigaon, Dhubri, Kokrajhar, Karbi Anglong, and Cachar exhibited greater dissimilarity from the control population in terms of morphological characteristics.Interestingly, the Ponkagaon (PGG) population of Golaghat district exhibited significant dissimilarity from the control population, while other Golaghat district populations maintained close similarities.

Cluster analysis
The dendrogram-based UPGMA algorithm analysis of morphological and seeding pattern data at the district level, led to identification of two major clusters in the resulting dendrogram.From the dendrogram based on morphological data, it was observed that the populations from Tinsukia and Dhemaji exhibited the close clustering with the control population (Fig. 12).

Biochemical analysis
The analysis of biochemical parameters in Assam lemon samples collected from various districts of Assam revealed substantial variation across the studied regions.Interestingly, we also observed a striking similarity between the biochemical parameters of the control populations and those from the districts of Dhemaji, Tinsukia, Lakhimpur, Dibrugarh, and Jorhat.

pH
The results of the pH analysis of different districts in Assam along with the control population revealed that samples from the Cachar district had the highest pH (2.78 ± 0.01), while the samples from Lakhimpur district showed the lowest (2.27 ± 0.02) (Fig. 13, Supplementary file 5).

% Juice content
The % juice content analysis of different districts in Assam and the control population showed significant variations in the juice content of the samples.The samples from Cachar district had the highest % juice content (57.61 ± 0.523%), while, the samples from Dhemaji district exhibited the lowest % juice content (32.13 ± 0.036%) (Fig. 13, Supplementary file 5).

Total soluble solids
The results of the total soluble solids content analysis for different districts in Assam along with the control population revealed that the samples from Nagaon district had the highest total soluble solids (8.9 ± 0.27°Brix), while Sonitpur had the lowest (5.4 ± 0.2°Brix) (Fig. 13, Supplementary file 5).

Citric acid
The results of the citric acid content analysis for different districts in Assam along with the control population revealed that Kokrajhar had the highest citric acid content (0.98 ± 0.064 g/ml), while Dhemaji had the lowest (0.08 ± 0.01 g/ml) (Fig. 13, Supplementary file 5).

Ascorbic acid
The results of the ascorbic acid content analysis for different districts in Assam along with the control population revealed that Dhubri had the highest ascorbic acid content (0.607 ± 0.011 mg/ml), while Kamrup Rural had the lowest (0.132 ± 0.026 mg/ml) (Fig. 13, Supplementary file 5).

Total sugar and reducing sugar
The results of the total sugar content esitimation for different districts in Assam, as well as the control population, revealed that Dhubri had the highest concentration of total sugar content (622.38 ± 5.646 µg/ml) and Cachar had the lowest (67.67 ± 0.97 µg/ml).Furthermore, results of the reducing sugar content analysis showed that Kokrajhar had the highest concentration of reducing sugar content (26.99 ± 0.6 µg/ml), while the lowest concentration was found in Lakhimpur (20.56 ± 0.6 µg/ml) (Fig. 13, Supplementary file 5).

Carotenoid content
The results of the carotenoid estimation for the various districts in Assam along with the control population showed that the higest concentration of carotenoid on the peel of Assam lemon fruit was observed in the Nalbari district (2.64 ± 0.001 mg/g), while the lowest concentration was observed in Cachar district (1.17 ± 0.0058 mg/g) (Fig. www.nature.com/scientificreports/

Chlorophyll content
The results of the chlorophyll content estimation for the various districts in Assam, as well as the control population showed that the highest concentration of Chlorophyll a, Chlorophyll b, and the total chlorophyll content on the peel of Assam lemon fruit was observed in Karimganj district (2.58 ± 0.0496 µg/g, 4.01 ± 0.012 µg/g, 6.59 ± 0.0958 µg/g), and the lowest concentration was observed in Bongaigaon district (0.97 ± 0.0027 µg/g, 1.99 ± 0.046 µg/g, 2.96 ± 0.0434 µg/g) respectively (Fig. 14, Supplementary file 5).

Pectin content
The results of pectin estimation for the various districts in Assam along with the control population revealed that Nalbari district had the highest concentration of pectin content on the peel of Assam lemon fruit (4.05 ± 0.282%), while Bongaigaon had the lowest concentration (2.14 ± 0.02%).On the other hand, Bongaigaon had the highest concentration of pectin content in the pulp of Assam lemon fruit (6.32 ± 0.02%), while Udalguri had the lowest (4.44 ± 0.572%) (Fig. 15, Supplementary file 5).

Equivalent weight
The result of equivalent weight estimation for the various districts in Assam along with the control population showed that Sonitpur had the highest concentration of equivalent weight on the peel of Assam lemon fruit (15.17 ± 0.25%), while Dima Hasao had the lowest concentration (7.08 ± 0.447%).On the other hand, Morigaon had the highest concentration of equivalent weight in the pulp of Assam lemon fruit (8.96 ± 0.7%), while Lakhimpur had the lowest (2.31 ± 0.129%) (Fig. 15, Supplementary file 5).

Methoxy content (MeO)
The result of methoxy content estimation for the various districts in Assam along with the control population showed that Nalbari had the highest concentration of methoxy content on the peel of Assam lemon fruit (0.24 ± 0.027%), while Morigaon had the lowest concentration (0.18 ± 0.02%).On the other hand, Dhubri had the highest concentration of methoxy content in the pulp of Assam lemon fruit (0.54 ± 0.5%), while Tinsukia had the lowest (0.18 ± 0.06%) (Fig. 15, Supplementary file 5).population showed that Dima Hasao had the highest concentration of anhydrounic acid content on the peel of Assam lemon fruit (3.64 ± 0.15%), while Morigaon had the lowest concentration (2.3 ± 0.07%).On the other hand, Bongaigaon had the highest concentration of anhydrounic acid content in the pulp of Assam lemon fruit (9.59 ± 0.08%), while Tinsukia had the lowest (3.64 ± 0.17%) (Fig. 15, Supplementary file 5).

Degree of esterification (DE)
The results of degree of esterification for the 22 selected different districts of Assam along with control population revealed that only Nalbari (52.61 ± 1.172%) district exhibited a high degree of esterification (> 50%) in peel samples.In pulp samples, Nalbari, Morigaon, and Nagaon districts displayed the highest esterification levels at 55.95 ± 1.528%, 55.67 ± 1.924%, and 51 ± 1.978%, respectively.These findings emphasize the notable esterification concentrations in these districts' peel and pulp samples compared to others, indicating potential variations in the composition of ester bonds in these regions (Fig. 15, Supplementary file 5).

Analysis of variance (ANOVA)
During the investigation of variation of Assam lemon from different populations of Assam based on morphological and biochemical characters, we observed that there is a significant variation among the population of selected districts (p < 0.05), indicating a variation in the different morphological characters including seeding pattern and biochemical traits.

Soil nutrient estimation
The soil nutrient assessment conducted for the current study revealed that the sampled populations exhibited a range from acidic to slightly alkaline.Most of the studied districts showed predominantly acidic soil, while populations from certain districts such as Lakhimpur, Golaghat, Sonitpur, Nagaon, Kamrup Metro, Kamrup Rural, Kokrajhar, and Dhubri displayed both acidic and alkaline pH levels.Interestingly, the control population and districts like Karbi Anglong, Dima Hasao, Cachar, and Karimganj showcased an alkaline soil nature.
The study also showed that the soil collected from all selected populations exhibited a significant presence of vital macro nutrients.The findings demonstrated that the soil in all populations of selected district contained a substantial amount (ranging from 0.505 to 1.5%) of Organic Carbon, except for the sites in Karbi Anglong, where the Organic Carbon concentration was comparatively lower (0.100-0.500%) compared to the populations of other districts.Moreover, the soil samples from the populations of Nagaon, and Dhubri along with the control population, displayed a mixed concentration of Organic Carbon, ranging from 0.100% to 1.500%.Furthermore, current study also revealed that the soil samples from all populations in the selected districts exhibit a notable concentration of phosphate, ranging from 22 to < 73 kg/ha, except for populations in Karbi Anglong district, which had phosphate levels > 22 kg/ha.In addition, the study findings revealed a significant presence of Potassium in the soil across most of the populations of selected districts, with concentrations ranging from 112 to 392 kg/ ha, except for the populations in Tinsukia, Dibrugarh, Dhemaji, Jorhat, Golaghat, Sonitpur, Morigaon, Kamrup Metro, and Kokrajhar.In these districts, the Potassium concentration (> 112 kg/ha) was relatively lower.On the other hand, the soil samples from Lakhimpur, Nagaon, Kamrup Rural, Nalbari, Bongaigaon, Dima Hasao, and Cachar displayed varying concentrations from low to high level of Potassium in the soil, ranging from > 112 to 392 kg/ha.Additionally, the results show that, all the populations in the selected districts, including the control population, displayed lower concentrations of Ammoniacal Nitrogen (15 kg/ha) and Nitrate Nitrogen (4-10 kg/ ha) in the soil (Supplementary file 6).
The study also revealed that in addition to macronutrients, the soil in different regions in the Assam is also abundant in various micronutrients (Copper, Zinc, Boron, Manganese, Iron, and Molybdenum) which plays a crucial role in the healthy growth and development of plants, even though they are required in smaller quantities compared to macronutrients.The findings demonstrated that the soil from all the selected districts contains a significant amount of Copper (ranging from 0.5 to > 2 ppm) except some populations of Cachar and Dhubri districts along with all the populations of Karimganj district, where Copper content in the soil is relatively lower (ranging from 0 to 0.5 ppm).It has also been noticed that the levels of Zinc in the soil samples are generally low, ranging from 0 to 0.5 ppm, except for specific areas in Tinsukia, Lakhimpur, and Cachar where the concentration of zinc was higher, ranging from 0.5 to 2 ppm.Again, Boron concentration in the soil samples exhibited significant variations across the sampled districts.Notably, Sonitpur, Nagaon, and Karbi Anglong districts displayed a significant presence (> 2 ppm) of Boron, whereas soil samples from the Cachar district and the control population showed considerably lower Boron levels (ranging from 1 to 2 ppm).In contrast, the soil samples of the remaining districts exhibited a diverse range of Boron concentrations, varying from 0.1 to > 2 ppm.A notable variation in Manganese concentration was observed among all the populations of selected districts, demonstrating high Manganese levels (> 4 ppm) in the accessions of Tinsukia, Dhemaji, Lakhimpur, Udalguri, Baksha, Nalbari, and Barpeta, while Sonitpur, Nagaon, Kokrajhar, and Karbi Anglong exhibited comparatively lower concentrations (ranging from 2 to 4 ppm) in contrast to the aforementioned districts in Assam.The concentration of Manganese were however found to be significantly low (0-2 ppm) in the populations of Dibrugarh, Jorhat, Golaghat, Morigaon, Kamrup Metro, Kamrup Rural, Bongaigaon, and the control population.Further, the soil samples from the populations of Dima Hasao, Cachar, Karimganj, and Dhubri displayed a diverse range of Manganese concentration, ranging from 0.2 to > 4 ppm.Additionally, the soil samples from Dhemaji, Lakhimpur, Dibrugarh, Jorhat, Golaghat, Sonitpur, Udalguri, Baksha, Nalbari, Barpeta, Karbi Anglong, Nagaon, Morigaon, Kamrup Metro, Kamrup Rural, and Karimganj along with the control population displayed a significant concentration (ranging from 3 to > 6 ppm) of Iron in the soil, whereas samples from all the populations in Dhubri were found to be significantly low in Iron concentration (ranging from 1 to 3 ppm).Further, the soil samples collected from Tinsukia, Dima Hasao, Cachar, and Kokrajhar exhibited a wide range of Iron concentrations, ranging from 1 to > 6 ppm.The concentration of Molybdenum however remained consistently low (ranging from 0 to 1 ppm) across all districts, including in the control population (Supplementary file 7).

Discussion
Morphological characters analysis plays a crucial role in the investigation of plants, enabling researchers to explore the vast array of variations in a plant species 37 .By closely examining the morphological characteristics of plants, valuable insights can be gained not only into the diverse traits and variations within the different plant species, cultivars, and varieties but also within the same cultivar and variety across different regions [38][39][40] .The morphological variation observed among Assam lemon populations from different districts of Assam provides valuable insights into the morphological variation within the cultivar.During the current study, we observed that there is a significant variation in several morphological characters including fruit weight, fruit length, fruit diameter, pulp to peel ratio and others among the 132 populations from the 22 districts along with the control, thus indicating variation in important fruit characters of Assam lemon fruits.The ANOVA analysis revealed a statistically significant difference (p < 0.05) among the populations, indicating a notable variation in morphological characters 41 .This finding highlights the presence of distinct morphological variations within the Assam lemon cultivar across different populations.Similar investigation was also observed on C. nobilis Lour 42 , Cynodon dactylon (L.) Pers. 43, C. reticulate Blanco 44 , which highlighted the differences in morphological traits across different populations.Interestingly, in the Upper Assam districts, excluding Golaghat, Assam lemon trees exhibit a flowering pattern wherein the prevalence of bisexual flowers surpasses that of unisexual flowers.Nevertheless, the proportion of unisexual flowers showed an increasing trend from Golaghat district towards Central, North, and Lower Assam, as well as Barak Valley, eventually reaching an almost equal concentration compared to bisexual flowers.This variation could be attributed to various factors, including environmental conditions, soil composition, and farmer practices specific to each region 7 .
The PCoA analysis of the population utilizing morphological characters provided compelling evidence of high morphological divergence among the Assam lemon populations.The results indicate presence of two distinct and well-defined groups that vary from each other in terms of their morphological traits.This emphasizes the significance of considering the morphological diversity within the Assam lemon cultivar, as it holds implications for genetic differentiation, adaptation, and potential breeding strategies 45 .These findings also open up avenues for further investigation into the underlying factors (such as environmental influences, genetic factors, or geographical factors) contributing to the observed morphological variations, thus can enhance our understanding of the complex dynamics within the Assam lemon populations 46 .Similar observations were also documented for UPGMA clustering analysis, where a total of 132 populations formed two distinct clusters.Notably, the second cluster displayed further subdivision into two sub-clusters.The UPGMA clustering analysis shows that the populations from Dhemaji and Tinsukia districts exhibit more morphological similarity with the control population www.nature.com/scientificreports/than other districts.Interestingly, in our previous reported work (Ahmed et al. in 2023), we observed similar outcomes while investigating the genetic diversity of Assam lemon using ISSR marker system where populations from Dhemaji and Tinsukia districts showed close resemblance with the control population 4 .This observation could be attributed to relatively fewer mutations occurring over time in these populations compared to other populations studied.In contrast, populations from Kokrajhar, Bongaigaon, Dhubri, Cachar, and Karimganj exhibited significant morphological differentiation from the control population.Similar observations has been made in few other species such as Cyclamen sp. 47, Cichorium sp. 48, C. Maxima (Burm.)Merr. 49.This implies that genetic variations or mutations could be one of the reasons for the observed variation in different characters in different regions of Assam 50 .
During the investigation of seed count among different populations in selected districts, we observed significant variation in the number of seeds.Specifically, the populations of Dhemaji, Tinsukia, Dibrugarh, Lakhimpur, and Jorhat, along with the control population, exhibited a seedless nature in their fruits which is its characteristic nature for which this cultivar was introduced and attained popularity 10 .However, the populations starting from Golaghat districts towards Central Assam, North Assam, Lower Assam and Barak Valley districts showed a mixed character, with some accessions having both forms and others were seedless fruits.Interestingly, the results of the seeding pattern resemble the variation in flowering nature.Assam lemon could be a self-incompatible cultivar having tendency towards natural hybridization [11][12][13] .
Additionally, the number of seeds found in Assam lemon fruits across different accessions was studied.The study observed variations in seed counts among different accessions, with specific patterns emerging in different districts with variations ranging from no seeds to more than 10 seeds.The variations in seed counts among different accessions of Assam lemon fruits, coupled with specific patterns observed in different districts, highlights the complexity of factors influencing seed pattern.It was previously reported that the environmental factors and genetic mutations act as a potential influencer on the seeding pattern of fruits 51 .Also, the variation in the soil nutrients in different districts of Assam may be a reason, which contributes to the differences in seeding patterns among the accessions in the current study 50 .Furthermore, the inherent propensity of Citrus species, including Assam lemon, for natural hybridization could potentially account for variations in seed formation and seed counts among different populations over time 52 .Given the contemporary emphasis on seedless fruit in response to consumer preferences, modern farmers and breeders are actively engaged in the development of diverse seedless fruit varieties 15 .The investigation into the variations in the seeding pattern of Assam lemon holds the potential to significantly impact cultivation techniques, aligning with consumer desires for enhanced fruit quality with seedlessness being a desired trait 53,54 .
Apart from the above-mentioned observations, variation in different biochemical parameters was found in the fruits of Assam lemon across the selected districts.A significant variation in pH concentration was observed in the samples from Karbi Anglong, Sonitpur, Baksha, Udalguri, Morigaon, Dhubri, and the Barak Valley districts (Dima Hasao, Cachar and Karimganj) where the pH was shown to be higher (> 2.5) as compared to other districts and the control population.The change in pH may be due to the variation in agro-environmental factors, such as soil nutrient composition, rainfall, and temperature, which can influence pH levels in fruits 55 .The current investigation also revealed that the samples from Nagaon, Kamrup Metro, and Kamrup Rural districts exhibited higher concentrations of TSS (≥ 8) compared to other districts and the control population, which is an important biochemical parameter used in various processing applications, such as juicing, canning, and making jams, jellies, and others 56 .The high TSS concentrations indicate an increased content of dissolved sugars, organic acids, and other soluble compounds in Assam lemon samples of Nagaon, Kamrup Metro, and Kamrup Rural districts 57 .Environmental conditions, including sunlight, rainfall, temperature, soil nutrients, and agricultural practices have been reported to affect TSS values in fruits 58 .These factors could be the reason for variation in TSS concentrations observed across different regions of Assam 58 .
Assam lemon is also rich in juice content as compared to other different lemon varieties due to its large size 5 .During the investigation we found that there is a significant variation in the % of juice content among different regions of Assam.All the districts along with the control population exhibited % juice content below 50%, except for Morigaon, Karbi Anglong, and the districts of the Barak Valley, which displayed a juice content exceeding 50%.Interestingly, the results also revealed that, although the populations from Dhemaji and Tinsukia districts exhibited resemblances with the control population in terms of morphological and other biochemical attributes, a notable difference emerged in terms of % juice content.Specifically, the population from the Dhemaji district displayed a lower % juice content (< 40%) at 32.13 ± 0.036% in comparison to both the control population and the Tinsukia district population (> 40%) at 41.24 ± 0.071% and 45.92 ± 0.92% respectively.The observed variations in juice content across different regions can be attributed to a range of regional factors, such as climate, soil composition, agricultural practices, and even genetic variations 59,60 .Further, during the current investigation, an interesting observation regarding the concentration of citric acid in Assam lemon across different districts was noted, it was observed that the districts of Dhemaji, Tinsukia, and Dibrugarh in Upper Assam and the control population had the lowest concentration of citric acid.However, as we moved towards Jorhat and Golaghat, the concentration of citric acid gradually increased.The districts in the Barak Valley exhibited the maximum concentration of citric acid.This variation in citric acid content suggests differences in the enzymatic activity responsible for citric acid synthesis during fruit development 61 .In addition to citric acid, we also examined the ascorbic acid content in the Assam lemon samples.Surprisingly, it was found that all the districts, along with the control population, had ascorbic acid content below 0.4 mg/ml, except for the samples from the Dhubri district, which displayed an ascorbic acid content of 0.607 mg/ml.The variation in acid content among the Assam lemon fruits could be attributed to environmental conditions, including temperature, sunlight exposure, rainfall, and other factors 62 .It has been reported that the ascorbic acid content of Citrus fruits is not stable and can vary due to enzymatic loss, where l-ascorbic acid is converted to 2-3-deoxy-l-gluconic acid 63 .Additionally, the utilization of ascorbic acid in metabolic processes might contribute to its decrease during the maturation process 64 .
On the other hand, it was observed that the fruits obtained from Dhemaji, Tinsukia, Dibrugarh, Jorhat, and Golaghat, as well as Kamrup Metro and Kamrup Rural districts, exhibited high level of TSS/TA compared to samples from other districts.Fruits from these regions displayed higher levels of TSS and a significant decrease in acid accumulation, which could be the contributing factors to the decline in the TSS/TA ratio 61 .Further, during the current investigation of Assam lemon fruit juice, a noteworthy finding emerged regarding the relationship between sugar concentration and pH, we observed a negative correlation between these two variables across most of the samples analyzed.This implies that as the sugar concentration increased, there was a corresponding decrease in the pH of the juice 65 .This intriguing pattern might be due to the accumulation of organic acids within the juice 66 .These organic acids not only impact the sugar concentration but also play a crucial role in determining the overall pH of the fruit juice 67 .Hence, the observed variations in sugar concentration may be attributed to the influence of these organic acids, which subsequently affect both the sugar concentration and the pH levels of the juice 66,67 .Additionally, while studying the carotenoid and chlorophyll content in samples of selected districts along with the samples from control population, a negative correlation between the carotenoid and chlorophyll content was observed.Low chlorophyll levels and high carotenoid levels may be favored by specific environmental conditions that promote carotenoid synthesis and accumulation 68 .The environmental factors such as light intensity, temperature, and nutrient availability may be the causes which influenced the pigment composition of Assam lemon peel 69 .
Pectin, a crucial ingredient used in various food products, including jams, jellies, and low-calorie foods, was found to be abundant in both the peel and pulp of the selected samples from different districts, including the control population 70 .However, it was noted that the pectin concentration was consistently high in the pulp compared to the peel.Interestingly, when comparing the pectin concentration among districts, it was found that samples from Upper Assam and the control population exhibited lower pectin concentrations compared to samples from other districts.Environmental conditions and genetic mutations might be the attributing factors to these variations, which have also been previously reported to influence pectin production 71,72 .Pectin can be categorized as either high-methoxy pectin or low-methoxy pectin based on its esterification extent 33 .Highmethoxy pectin is known for its sensitivity to acid conditions and typically requires a substantial amount of sugar to function as a thickening and gelling agent 73 .On the other hand, low-methoxy pectin has gained prominence in the food industry, particularly for jam production, as it can form a gel with less reliance on sugar 73 .To ensure the purity of the extracted pectin, we also evaluated the anhydrounic acid content in both the peel and pulp of Assam lemon samples 33 .Notably, the pulp displayed the highest anhydrounic acid content, followed by the peel.Findings from the current study indicated that samples from the control population, Upper Assam, Barak Valley, Bogaigaon, Kokrajhar, and Dhubri districts have highest pectin purity compared to samples from other districts.The present investigation also delved into the esterification level of pectin, specifically focusing on the concentration of methylesters, which plays a significant role in determining the applicability of pectin in various food industries 74 .Notably, the peel samples from Nalbari and the pulp samples from Morigaon and Nalbari exhibited high concentrations of methylesters, indicating the presence of high-methoxy pectin.In contrast, the samples from all other districts, including the control population, displayed low concentrations of methylesters, suggesting the prevalence of low-methoxy pectin.Factors such as climate, soil composition, and agricultural practices specific to each region may contribute to these variations 55 .These factors may collectively influence the physiological characteristics of Assam lemon trees, leading to variations in fruit development and juice production 45,75 .
The soil composition and nutrient availability play a pivotal role in shaping the morphological characters of plants, including various fruit traits such as length, diameter, peel thickness, seed number, and more 76 .During the current investigation, soil nutrient analysis from 132 populations revealed a significant variation not only in the nutrient content but also in the soil nature itself.This variation in soil characteristics is likely to be a crucial factor contributing to the observed variations in different morphological characters of Assam lemon within the selected populations 77 .It's important to note that soil fertility and nutrient content can vary widely based on geographical and environmental factors.Therefore, regular soil testing and analysis are essential for farmers to make informed decisions regarding nutrient management and crop production.
By catering to demands for high yield, substantial juice content, and the absence of seeds, the outcomes of this study can play a pivotal role in guiding cultivation practices and meeting the evolving expectations of consumers 54 .Thus, the findings of this study have practical implications for growers and breeders as they can select suitable populations with desired morphological characters for commercial cultivation and breeding programs.

Conclusion
The present investigation underscores distinct variations in morphological, seeding patterns, and biochemical traits among Assam lemon accessions, with a notable affinity of Upper Assam districts to the control.Remarkably, the Upper Assam population demonstrates a noteworthy resemblance to the control, particularly in terms of its flowering characteristics.This includes the presence of both bisexual and unisexual flowers, with less concentration of unisexual flowers.Moreover, the seed availability pattern in these populations is notable, featuring the development of seedless Assam lemon fruits.In contrast, populations from other selected districts exhibited both bisexual and unisexual flowers with nearly equal concentrations.Additionally, these populations harbour both seeded and seedless fruits.The identified variation in the seeding pattern of Assam lemon holds promising potential to significantly influence cultivation practices, meeting consumer preferences for seedless fruits and improved quality.Further, biochemical analyses unveiled significant variation across districts, although populations from Dhemaji, Tinsukia, Lakhimpur, Dibrugarh, and Jorhat demonstrated similarity with the control.These insights contribute to our understanding of the cultivar's diversity and hold implications for future breeding programs.By catering to the growing demands for high yield, substantial juice content, nutritionally rich and www.nature.com/scientificreports/ the of seeds, the findings of this study carry substantial importance in shaping cultivation practices.Thus, the findings of this study have practical implications for growers and breeders as they can select suitable populations with desired morphological characters for commercial cultivation and breeding programs. Vol

Figure 1 .
Figure 1.Map of Assam, India showing the study area, prepared using ArcGIS software (version 10.2).

Figure 2 .
Figure 2. Map of Assam showing the geographic locations of the sampled population in Upper Assam, prepared using ArcGIS software (version 10.2).

Figure 3 .
Figure 3. Map of Assam showing the geographic locations of the sampled population in North Assam, prepared using ArcGIS software (version 10.2).

Figure 4 .
Figure 4. Map of Assam showing the geographic locations of the sampled population in Central Assam, prepared using ArcGIS software (version 10.2).

Figure 5 .
Figure 5. Map of Assam showing the geographic locations of the sampled population in Barak Valley, prepared using ArcGIS software (version 10.2).

Figure 6 .
Figure 6.Map of Assam showing the geographic locations of the sampled population in Lower Assam, (prepared using ArcGIS software (version 10.2).

Figure 13 .
Figure 13.Biochemical characteristic of Assam lemon fruit juice across different districts of Assam; (a): pH, (b): TSS, (c): % Juice Content, (d): Citric Acid, (e): Ascorbic Acid, (f): TSS/TA, (g): Total Sugar, (h): Reducing Sugar.Data represent mean values (column bars) with standard deviation of n = 3 biological replicates represented as vertical lines on the column bars.Different letters on top of the bars represent significant differences between samples at subset for alpha = 0.05, based on Duncan Multiple Range Test (DMRT) test.

Figure 14 .
Figure 14.Biochemical characteristic of Assam lemon fruit juice across different districts of Assam; (a): Carotenoid Content, (b): Chlorophyll Content.Data represent mean values (column bars) with standard deviation of n = 3 biological replicates represented as vertical lines on the column bars.Different letters on top of the bars represent significant differences between samples at subset for alpha = 0.05, based on Duncan Multiple Range Test (DMRT) test.